Vitamin E (tocopherol) is the major fat-soluble antioxidant in humans, and serves a critical function in protection against a plethora of oxidative-stress-related pathologies. However, surprisingly little is known about the molecular mechanisms that regulate the levels and activities of the vitamin in the body. The hepatic alpha tocopherol transfer protein (TTP) is a critical regulator of vitamin E status; humans carrying mutated TTP alleles develop severe vitamin E deficiency, accompanied by neuronal degeneration. TTP is thought to function as a 'gatekeeper' in the liver, where it selectively retains the most biologically active form of vitamin E (RRR-1-tocopherol), and 'directs' it to plasma lipoproteins that deliver the vitamin to peripheral cells. Here, we propose to continue our in vivo and in vitro studies on TTP with the overall goal of understanding the molecular bases for the function of the protein in mediating the effects of vitamin E, both in health and in disease. We will use our combined expertise in synthetic chemistry, biophysics, biochemistry and cell-biology to address four specific aims: 1)- to characterize the steps involved in the transport of vitamin E in hepatocytes, and the exact role of TTP in stimulating this process. Using novel fluorescent analogs of vitamin E, we will monitor the intracellular trafficking of the vitamin in cultured hepatocytes. We will then use molecular- and cell-biology approaches to define the steps that TTP catalyzes, and the molecular mechanisms that underlie these events. 2)- To determine the factors and mechanisms that regulate the expression and degradation of TTP, and the roles that vitamin E status and / or oxidative stress play in these processes. 3)- To examine the functions of TTP in neurons, where the protein is expressed in significant levels, and which are the primary site of damage due to vitamin E deficiency. 4)- To understand the molecular mechanisms by which TTP catalyzes the inter-membrane transfer of vitamin E in vitro. Studies proposed in this application will provide critical information that will improve our understanding of vitamin E function as a mediator of human health. PUBLIC HEALTH RELEVANCE: Vitamin E is an essential nutrient that is required for protection of all cells from oxidative damage. TTP is the only known protein that specifically regulates the levels of vitamin E in the body. We propose to determine what exactly TTP does in liver cells, and how mutations in TTP that are found in humans affect its activity.